1. Field of the Invention
The present invention relates generally to the cost-effective recovery of saponins and sapogenins from plant material. In particular, the invention relates to the recovery of saponins and sapogenins from Chenopodium quinoa (Quinoa) (Chenopodiaceae) grain, bran and other plant parts in substantially pure and commercially useful forms.
2. Description of Related Art
The high levels of saponins found in certain plants has long been thought to be responsible for the medicinal effects of some of these plants (Waller, G. R. and K. Yamasaki, Saponins used in Traditional and Modern Medicine, Advances in Experimental Medicine and Biology, Vol.404, 1996, New York: Plenum Press). The presence of high levels of saponins in the seeds of plants such as Quinoa (Chenopodium quinoa) has restricted the use of the human consumption of this grain.
Quinoa originates from the Andes region of South America where it was a staple grain in pre-Spanish Conquest times. Traditional use declined after the Spanish Conquest (Galwey, N. W., et al., Food Sci. Nutr., 42F:245, 1990) and cultivation and use of the grain was not widespread until a recent revival due to western interest in this crop as a high lysine, high protein grain for human consumption (De Bruin, A., J. Food Sci., 26:872,1964). The principal obstacle to wider human consumption of this grain has and continues to be the bitter taste of the saponin content of the grain. These saponins have been shown to be anti-nutritive in animal studies (Gee, J. M., et al., J. Sci. Food Agric., 63:201, 1993). In traditional use, the saponin content of the grain was reduced to acceptable levels by washing the grain in running water.
Since the revival of interest in Quinoa, a number of attempts have been made to devise practical methods to reduce the saponin content (Amaya-Farfan, J., et al., Removal of saponins from quinoa (Chenopodium quinoa Willd) grain by milling, 5th Int. Congr. Food Sci. Technol., Kyoto, Japan (1978); Gee, J. M., et al., J. Sci. Food Agric., 63:201,1993; Reichert, R. D., et al., Cereal Chem. 63:471, 1986; Galwey, N. W., et al., Food Sci. Nutr. 42F:245, 1990; Rios, M. L. T., et al., Arch Latinoamer Nutr. 28:253, 1978; Ridout, C. L., et al., J. Sci. Food Agric. 54:165, 1991), including combinations of milling and washing. In all cases the saponin rich fraction was considered to be a waste product and was discarded.
The recent interest in nutraceuticals and the medicinal properties of plants has resulted in studies that have attributed the biological activity of many of these plants to their saponin content. Many interesting physiological and pharmacological effects have been attributed to saponins and/or the corresponding sapogenins including reduction of serum cholesterol (Price et al., CRC Crit. Rev. Food Sci. Nutr. 26:27 (1987)), inhibition of alcohol absorption (Yoshikawa, M. and J. Yamahara (1996), In Saponins used in Traditional and Modern Medicine, Edited by G. R. Waller and K. Yamasaki. pp.207-218. New York, Plenum Press, Vol. 404), inhibition of glucose absorption (Matsuda, H., et al., Biol. Pharmac. Bull. 20:717, 1997), facilitation of transdermal absorption and intestinal absorption of drugs (Gee, et al. Toxic. in Vitro 3:85 (1989)), hypoglycaemic and anti-inflammatory effects (Honda, T., et al., Bioorganic Med. Chem. Lett. 7:1623,1997). Recently studies have shown that novel derivatives of the sapogenin oleanolic acid have potentially valuable pharmacological properties (Finlay, H. J., et al., Bioorganic Med. Chem. Lett. 7:1769,1997).
Saponins have been known to have adjuvant activity since the 1920""s (Sjxc3x6lander and Cox, Adv. Drug Delivery Rev. 34:321 (1998)), and a significant body of research has been conducted to explore these properties, particularly with saponin containing extracts of Quillaia saponaria. Kensil (Kensil, et al., J. Immunol. 146:431(1991)) has demonstrated that a need exists for a substantially pure saponin that can be used as an adjuvant in relatively low quantities with low toxicity and side effects. Estrada et al. U.S. Pat. No. 5,688,772 teaches that all quinoa saponins obtained by water extraction are equivalent and active as immunological adjuvants.
In spite of this interest, only a very limited number of purified saponins and sapogenins are commercially available and practical procedures for large scale quantitative and qualitative recovery of highly purified saponins and sapogenins are lacking in spite of numerous publications describing analytical and laboratory scale procedures. The lack of suitable practical extraction and purification methods is also reflected in the relatively high cost of those compounds that are available.
Traditionally, the saponin content in plant extracts has been determined by bioassay or by GLC analysis of the sapogenins derived by hydrolysis of the saponins (Ridout et al., J. Sci. Food Agric. 54:165 (1991)). GLC analysis in particular requires extensive clean up and hydrolysis prior to derivatization and analysis and this is reflected in the prior art for the extraction and purification of these compounds. Recent developments in HPLC analysis in our laboratories have indicated that, in the case of quinoa saponins, the GLC approach of extensive purification does not give quantitative or qualitative recovery of the naturally occurring saponins.
The chemical nature of the saponins found in quinoa has been the subject of several investigations (Mizui, F., et al. Chem. Pharm. Bull., 38:375 (1990)); however, the procedures used in these investigations for the recovery of the saponins is not practical and applicable for commercial scale production. The studies of Mizui et al. (Mizui, F., et al. Chem. Pharm. Bull., 38:375 (1990)) and others have shown that the saponins found in quinoa are of the triterpene type.
The prior art for isolation of saponins from quinoa falls into two categories: a) an aqueous extraction route typically as described in Estrada et al., 1997, U.S. Pat. Nos. 5,597,807 and 5,688,772; and b) a more traditional hot alcohol solvent (Mizui, F., et al. Chem. Pharm. Bull., 36:1415 (1988); Mizui, F., et al. Chem. Pharm. Bull., 38:375 (1990)). Surprisingly, the inventors of the present invention, have determined that neither the aqueous extraction route nor the hot alcohol extraction route are particularly efficient in recovery of quinoa saponins from bran, nor do either solvent extract the saponins from quinoa seed or bran on a qualitative basis. Estrada et al. U.S. Pat. No. 5,688,772 teaches that water extracts of quinoa (10 g of hulls extracted by 2xc3x97100 mL of water) contain all or most of the saponins present in quinoa.
Surprisingly, while the aqueous extraction method provides an extract with similar saponin profiles to that now known to be present in quinoa grain or bran, the yield was only 20% of that obtained by the process of the present invention. Mizui et al. [Mizui, F., et al. Chem. Pharm. Bull., 36:1415(1988); Mizui, F., et al. Chem. Pharm. Bull., 38:375 (1990)] demonstrated that saponins could be extracted from quinoa bran with hot methanol with yields of between 20 and 25% depending upon whether a subsequent hot 50% methanol extract was employed. These yields are significantly less than those achieved using the process of the present invention.
Surprisingly, the inventors have also shown that extraction with pure alcohols is highly selective in that methanol preferentially extracts only one of the three main saponins.
Surprising also is the very low yield of saponins obtained by this approach which is reflected not only in the results achieved by the present invention, but also in the very low yield of saponins obtained by Mizui et al. (1.66%).
The prior art for the purification of quinoa saponins is lacking in any specific details that would allow commercial scale production of these compounds. For example, Estrada et al., 1997 U.S. Pat. Nos. 5,597,807 and 5,688,772, and Estrada et al. Conun. Immun. Microbial and Infect. Dis. 21:225 (1998), teaches that quinoa saponins can be purified by dialysis against water or phosphate buffered saline (PBS) pH 7.2; however, since the inventors of the present invention have subsequently demonstrated that the aqueous extraction approach is not particularly efficient (10% of the weight of bran recovered as a saponin extract), and not the preferred extraction method, an alternative approach is required. Surprisingly, the inventors have also observed that dialysis of aqueous quinoa saponin extracts is a relatively inefficient purification step with only modest reduction in nonsaponin components. The approach of Mizui et al. [Mizui, F., et al. Chem. Pharm. Bull., 36:1415 (1988); Mizui, F., et al. Chem. Pharm. Bull., 38:375 (1990)] is complicated, involving multiple chromatography steps with low yields.
Prior art for the extraction of triterpene saponins from whole plants or seeds also includes a number of different solvent systems including acetone, diethyl-ether and ethyl acetate (Tanaka, O and Yata, N., U.S. Pat. No. 4,501,734). Surprisingly, the inventors of the present invention have found that only a narrow range of solvents can effectively extract saponins from quinoa bran.
Prior art for the purification of triterpene saponins from other whole plants or seeds includes a number of different procedures. For example, Combier, H. et al., (U.S. Pat. No. 4,335,113), teach that saponins can be extracted from an aqueous solution by n-butanol/benzene mixture after prior extraction with ethyl acetate. These researchers also teach that saponins can be purified by successive chromatography on silica gel (CHCl3:CH3OH:H2O; 65/25/10 to 50/40/10 v/v/v) and reversed phase liquid chromatography (RP-HPLC) (CH3OH:H2O). Tanaka, O and Yata, N. (U.S. Pat. No. 4,501,734) also teaches that saponins can be purified by sequential extract of an alcoholic extract with n-hexane and ethyl acetate, prior to extraction of the saponins into n-butanol followed by column chromatography on silica gel chromatography using combinations of ethyl acetate, chloroform, n-butanol, methanol, ethanol and water as eluants. Surprisingly, but not unexpectedly since (Tanaka, O and Yata, N. U.S. Pat. No. 4,501,734) reported that ethyl acetate could be used to extract saponins, the inventors have observed that ethyl acetate will extract some saponins from an aqueous solution of quinoa saponins. The impracticability of such an approach is illustrated by recovery of only 5% of the n-butanol fraction as purified saponins. Combier, H. et al., (U.S. Pat. No. 4,335,113) also teach that the saponin containing butylic soluble component of the hot methanolic extract of Chrysanthellum sp. can be treated with activated charcoal, however no effect of the activated charcoal on saponin content or purity was shown.
Surprisingly the inventors have repeatedly observed that n-butanol and other higher alcohols are selective in their ability to recover quinoa saponins from the aqueous extracts derived from quinoa and also that recovery of saponins in the water immiscible higher alcohol fraction is surprisingly inefficient.
Kensil et al. (WO 88/09336, U.S. Pat. No. 5,057,540, Oct. 15, 1991) describe a process in which Quillaja saponaria bark is extracted with water, dialysed and the resulting extract lyophilized. The saponins are solubilized from the resulting powder with methanol and subjected to silica gel chromatography and/or RP-HPLC. This process was however only conducted on samples of less than 2 grams. When applied to quinoa extracts the inventors have observed little value in the dialysis step and observed that relatively little of the quinoa saponin can be resolubilized in methanol. Dorn (U.S. Pat. No. 3,883,425, May 13,1975) describes a process in which aqueous commercial saponin preparations (5 to 40% w/v) are freed from antibacterial toxins (undefined low molecular weight compounds) by passage over ultrafiltration membranes with molecular weight exclusion limits ranging from 500 to 30,000 in which losses of saponins to the permeate are claimed to be 5% or less except for the 30,000 MWCO membrane in which case the losses were reported to be 7.5%. Surprisingly the inventors find that crude quinoa saponin powders obtained from aqueous alcohol extracts of quinoa bran do not readily dissolve in water to form high molecular weight micelles. Not unexpectedly, the presence of even a small amount of alcohol appears to prevent the formation of high molecular weight micelles.
When the prior art was applied to the extraction and purification of saponins from quinoa or quinoa bran, the yield of total saponins was either significantly lower than could be achieved by the technology described herein and/or resulted in only selective recovery of some of the saponins present.
Sapogenins
Approaches for the isolation and purification of sapogenins have also been described in prior art including the isolation of oleanolic acid from Diospyros kaki (Liu, Y. U.S. Pat. No. 5,086,043, Feb. 4, 1992), however these methods are not applicable to quinoa seed and quinoa bran which contain three different sapogenins (Oleanolic acid, hederagenin and phytolaccagenic acid).
Loken (U.S. Pat No. 3,895999, July 22, 1975, U.S. Pat. No. 3,510,400) teaches that the preferred hydrolysis conditions for the generation of sapogenins is hydrolysis of an aqueous extract in the pH range 1.0 to 2.5 at temperatures in the range of 110xc2x0-145xc2x0 C. followed by partial neutralization to pH 5-6. Surprisingly the inventors have determined that by conducting the hydrolysis in an aqueous alcohol environment the temperatures required are reduced to the boiling point of the aqueous alcohol solvent, typically 75-80xc2x0 C., without significantly extending the hydrolysis time, thus representing a significant reduction in cost and removing the requirement for a pressure vessel. Prior art for the hydrolysis of triterpene sapogenins to their corresponding sapogenins describes only analytical approaches, typically 1.5% H2SO4 at 70xc2x0 C. for 20 h (Solvent not defined).
Other traditional approaches to recovery of sapogenins include that described by Rajasekaran, M., et al., J. Ethanopharmacol 24:115,1988, who teach that oleanolic acid can be recovered from flowers of Eugenia jambolana by refluxing with 95% ethanol and chromatography of the benzene soluble portion of the extract on silica gel eluted with chloroform/methanol. Umehara, K., et al., Chem. Pharm. Bull. 40:401, 1992, also describe a similar process involving methanol extraction of cloves (Syzygium aromaticum) and subsequent solvent partitioning and silica gel chromatography. In this procedure when solvent partitioning is used to affect separation, the sapogenins of interest were distributed between the benzene and the methanol partitions. This is clearly not desirable in a commercial recovery process. It is also not apparent from their publication what form the oleanolic acid occurs in the clove (saponin or sapogenin). Singh, G. B., et al., J. Pharm. Pharmacol. 44:456,1992, describe a process for recovery of oleanolic acid from Luffa cyllndrica seed in which the saponins were extracted with methanol and oleanolic acid liberated by acid hydrolysis. The crude oleanolic acid was washed exhaustively with acetone and pure oleanolic acid recovered by recrystallization from ethanol. These approaches are not practical for large scale production of sapogenins and are impractical for plants that contain mixtures of sapogenins.
There is therefore a need for a simple practical process for preparation and recovery of highly purified saponins and sapogenins from quinoa that is effective on a commercial scale.
Definitions
It is felt that the following definitions may assist with the understanding of the description of the present invention.
By xe2x80x9csaponinxe2x80x9d is meant a compound consisting of a triterpenoid of oleanane structure and one or more glycosides, the glycosides being bound to the triterpenoid at the 3 position and/or at the 28 position.
The term xe2x80x9cglycosidexe2x80x9d is intended to mean all sugars including glucose found naturally in quinoa including arabinose, glucose, galactose, xylose and glucuronic acid.
By xe2x80x9csapogeninxe2x80x9d is meant the triterpenoid alone without glycosides attached at either the 3 or the 28 position.
By xe2x80x9cquinoa branxe2x80x9d is meant the bran obtained in a commercial mill used to de-bran quinoa for human consumption.
Some standard abbreviations used in connection with the present invention include:
HPLCxe2x80x94high pressure liquid chromatography (suitable apparatus for this includes (1) Waters Corporation Model 2690 Separations module (Alliance) with a 996 PDA detector; and (2) Hewlett Packard Model 1090 hplc system);
TFAxe2x80x94trifluoroacetic acid;
ELSDxe2x80x94evaporative light scattering detector;
GLCxe2x80x94gas liquid chromatography;
RPxe2x80x94reversed phase;
MWCOxe2x80x94molecular weight cut-off; and
SPExe2x80x94solid phase extraction (suitable apparatus includes Pharmacia Model Process Stack Column PS 370, pump Spectra/Chrom Macroflow pump (head model 7090-42), resin Waters preparative C18 125A bulk packing 55-102 xcexcm).
An object of the present invention is to provide a process of extraction of saponins (and ultimately sapogenins) from quinoa that can be operated on a commercial basis.
According to one aspect the present invention, there is provided a process for commercial extraction of saponins from quinoa, comprising: contacting a saponin-containing part of a quinoa plant with an aqueous alcohol solution containing an alcohol selected from the group consisting of methanol and ethanol to form a saponin-containing solution and an extracted solid residue, removing the alcohol from the saponin-containing solution to leave a saponin-containing aqueous solution, and evaporating water from the saponin-containing aqueous solution to produce a saponin-containing product.
According to another aspect of the invention, there is provided a process of producing sapogenins from corresponding saponins obtained by extraction from a quinoa plant, comprising: obtaining a solution of saponins in an aqueous alcohol, adding a strong (preferably 1-3.5 N) acid to the solution to hydrolyze the saponins to form corresponding sapogenins that precipitate out of the solution as a precipitate, recovering the precipitate, and decolorizing the precipitate by forming a slurry of the precipitate with a solution of an aqueous base to form a decolorized sapogenin product.
The aqueous alcohol solution used for the initial extraction of the saponins from the plant parts preferably contains 40 to 80% alcohol by volume. Amounts above and below this range tend to result in the extraction of significantly less of the saponins and tends to result in an uneven pattern of extraction of the different individual saponins present in the plant parts. In.fact, the range of 50 to 75% alcohol is more preferred for these reasons, and the most preferred range is 50 to 60%. The optimum amount is about 50% alcohol by volume.
The ratio of extraction liquid to plant parts (e.g. bran) used for the extraction is preferably at least 8:1 v/w, more preferably at least 10:1. At smaller ratios of liquid to solid, the mixture may be too stiff to stir. When bran or quinoa flour are employed as the starting materials for extraction, there is a particular tendency for the mixture to become too thick and stiff as the plant material absorbs liquid and tends to swell. In these cases, the 10:1 ratio is the preferred minimum. As for maximum ratios of liquid to solid, there is generally no advantage in using more than 30:1 or even 15:1. As more liquid is added, more has to be removed in subsequent steps, but there is usually no significant increase in rates of extraction. The preferred ratio is therefore 10-15:1.
After extraction, the alcohol component is removed from the extraction liquor. While this can be done by any means, it is most desirable to use flash evaporation. Flash evaporation is a technique known in preparative chemistry for the rapid removal of a volatile component from a liquid mixture. The volatile liquid is removed from solution by rapid conversion to a vapor phase by creating a thin film of the solution over a large surface area under reduced pressure often accompanied by an increase of temperature of the solution above ambient but less than the boiling point of the solution at atmospheric pressure. The actual thickness of the film and the area over which it is applied is chosen to provide optimum evaporation and ease of use, but evaporation may be substantially instantaneous (hence the name xe2x80x9cflashxe2x80x9d evaporation). Flash evaporation avoids the prolonged use of high temperatures that may degrade the intended product and has the ability to remove almost all of the alcohol component (which makes the remaining solution suitable for the preferred practice of spray drying employed in the next step. The alcohol may be recovered from this step and re-used in the extraction process.
For the removal of water from the extraction liquor, spray drying is preferred, although other techniques could be employed. Spray drying is a known technique regularly used in preparative chemistry and the food processing industry in which a fine spray of droplets of the liquid is introduced into a moving gas (usually air) flow to cause loss of moisture from the droplets). The gas is often heated, e.g. it may have an inlet temperature in the range of 80-150xc2x0 C. and an outlet temperature that is lower, typically 50-100xc2x0 C. (actual temperatures are usually machine-dependent and are adjusted to achieve optimum results). Spray drying is rapid and again may avoid the prolonged exposure of the product to high temperatures. The dry product resulting from spray drying is obtained in the form of a fine powder that is easy to collect, manipulate, store and re-dissolve.
The extraction process of the invention is preferably carried out on a commercial variety or cultivar of quinoa using a dry (non-green) part, and most preferably a bran product obtained by dry milling to remove seed coats from commercial quinoa grain. Quinoa bran is commercially available and inexpensive (virtually a waste product) resulting from the treatment of quinoa seed to form a consumable flour. Quinoa bran is rich in saponins (the bran contains approximately 50 times more saponin than could be recovered by washing the whole grain or extracting ground seed) and can be obtained by dry milling of high saponin content quinoa. However, if desired, other (preferably not green) parts of the quinoa plant may be used as starting materials for the saponin extraction, e.g. whole seeds, ground seeds, seed coats or quinoa flour.
The saponin content of the quinoa starting material (e.g. bran) and the ensuing fractions can be monitored, for example, by HPLC analysis of a filtered 50% (v/v) ethanol or methanol extract of the bran by chromatography on C-8 or C-18 RP columns eluted with a 0.05% Trifluoroacetic acid (v/v) (TFA) in water:methanol gradient, or a 0.05% TFA in water:acetonitrile gradient. Saponins in the samples are detected by Evaporative Light Scattering Detection (ELSD) using, for example, Model PL-EMD 960 from Polymer Laboratories (settings: atten 1; air temp. 90xc2x0 C.; air flow 3.7 l/m). Acetic acid (1%) can be used in place of TFA and chromatographic separation can be achieved by isocratic elution. The sapogenin content of extracts and samples derived by hydrolysis can also be determined using the same chromatographic procedure.
A substantially enriched dry saponin fraction is obtained by extraction of the bran with 50% v/v aqueous alcohol (methanol or ethanol), evaporation of the alcohol (methanol or ethanol) and spray drying of the concentrate to give a powder. Surprisingly, extraction of bran or seed with aqueous alcohol yields an extract that contains more than twice the amount of saponin than extraction with either water or pure alcohols alone.
Also surprisingly, both water and alcohol, when used individually, differentially extract saponins (of which there are usually three main kinds) from the bran with the result that the saponin composition of the pure alcohol extract is substantially different from the aqueous alcohol or aqueous extracts. In the product of the process of the present invention, the saponin profile of the bran extract is substantially the same as that of the quinoa starting material, e.g. quinoa seed, which leads to greater extraction efficiency and yield.
Surprisingly the inventors have also observed that other extracting solvents and conditions described in the prior art as being suitable for extraction of saponins are largely ineffective in removing saponins from quinoa bran or whole seed.
The saponin content of the aqueous alcohol extract can be further increased by passage over a 1000 MWCO spiral wound ultrafiltration membrane (e.g. an Amicon Model S3Y1 spiral wound ultrafiltration cartridge) without significant alteration to or loss of the saponin composition. This concentrated saponin fraction where the saponin content is in the range of 85-90%, can then be further purified in a liquid state or reduced to a dry state. Individual saponins are recovered by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC (e.g. using Waters Corporation Model Prep 4000 with a 486 tunable wavelength detector). Alternatively, the aqueous alcohol extract containing saponins can be fractionated directly by a combination of reversed-phase solid phase extraction and preparative reversed-phase HPLC, however this is less efficient in the absence of the membrane pretreatment.
A concentrated solution of the sapogenins can be obtained by acid hydrolysis, for example using 450 mL concentrated HCl per 3 L of the aqueous alcohol extract (e.g. 50% v/v ethanol) under reflux (e.g. for 6 hours). The hydrolysate is allowed to cool resulting in the formation of a precipitate which is recovered by filtration. The precipitate is slurried in water (e.g 2 L) and the resulting slurry is adjusted preferably to pH 10 with a base (NaOH). The sapogenins precipitate from the basic solution as off-white crystals and are recovered by filtration. The resulting crystalline precipitate is washed with dilute acid (e.g. 2 L of 1.0 N) and distilled water until the effluent is clear. Surprisingly, the precipitate is essentially free from coloured impurities, where as precipitated sapogenins obtained under neutral or acidic conditions are dark brown in colour. The precipitate containing the sapogenins may then be air-dried and can be further refined by recrystallization.
The individual sapogenins may be recovered from this mixture, e.g. by preparative HPLC using reversed-phase adsorbents. The purification can also be achieved on a large scale by selective desorption from a reversed-phase solid-phase extraction cartridge eluted with a step gradient of aqueous methanol. Preparative HPLC and systems such as simulated moving bed chromatography are frequently in commercial use for recovery of high value solutes from solutions. The sapogenins may be further purified by recrystallization from hot 95% alcohol.